Methods of inducing ovulation using a non-polypeptide camp level modulator

ABSTRACT

The present invention relates to methods of inducing ovulation in a female host comprising the administration of a non-polypeptide cyclic adenosine monophosphate (cAMP) level modulator to female host. In another aspect, the invention provides for specific administration of the phosphodiesterase inhibitor prior to the luteal phase of the host&#39;s ovulatory cycle. Preferred non-polypeptide cAMP level modulator include phosphodiesterase inhibitors, particularly inhibitors of phosphodiesterase 4 isoforms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the 371 National Stage of International ApplicationNo. PCT/EP01/14730, filed Dec. 14, 2001.

FIELD OF THE INVENTION

The present invention relates to methods of enhancing fertility in afemale host by inducing ovulation with the administration of anon-polypeptide modulator of cAMP levels.

BACKGROUND OF THE INVENTION

Ovulation is the process where an ovum or ova are released from theovaries. The timing of ovulation within the menstrual cycle is offoremost importance for fertilization. It is well recognized thatfollicles acquire the ability to ovulate following growth and maturationstimulated by the pituitary gonadotropins. Follicle stimulating hormone(FSH) is predominantly responsible for follicular growth and luteinizinghormone (LH) stimulates ovulation. This coordinated process ofgonadotropin-stimulated maturation of the follicle ensures delivery of acompetent ova at ovulation. The adequately prepared ovum is thenavailable for fertilization by sperm within hours after ovulation.

Ovulation is a finely timed process that is driven by pituitarygonadotropin stimulation of the ovary, and modified by the growth andbiochemical (e.g., steroidogenic, inhibin secretion, etc.) response offollicles to the gonadotropin stimulation. During the normal menstrualcycle in women these hormones exhibit cyclic patterns. The menstrualcycle can be functionally divided into three phases; the follicular, theovulatory and luteal phases. The follicular period begins at the end ofthe luteal phase of the preceding non-conceptive cycle, prior to orcoincident with the onset of menses. The cycle starts with a transientrise in blood levels of FSH that stimulates development of a cohort ofovarian follicles. The size of the follicles recruited to grow is about5 mm in diameter. In a natural menstrual cycle, usually one large ordominant follicle is established during the follicular phase, and it iscommitted to growth to maturation. In humans, the size of the folliclethat is considered ready to ovulate is about 15 mm or more in diameter.

The second critical event that occurs in the ovary during the follicularphase is that granulosa cells within the ovarian follicles acquirereceptors for LH and become increasingly responsive to LH. Secretion ofestradiol and estrone from the ovary increases slowly at first, inparallel to the increasing diameter of the follicle and sensitivity ofthe follicle to LH. The relatively rising levels of estrogen and inhibincause inhibition of gonadotropin releasing hormone (GnRH) secretion fromthe hypothalamus and gonadotropin secretion from the pituitary. Estrogenproduction reaches a maximum on the day before the LH peak and theneuroendocrine response to increased estrogen and gradually increasingconcentrations of progesterone is the preovulatory release ofgonadotropins which is discussed below.

During the ovulatory phase there is a change in the neuroendocrineresponse to estrogen and progesterone. At this point in the cycle,elevated estrogen elicits a preovulatory surge in serum FSH and LHlevels, due to a positive feedback on the hypothalamus, estrogen nowstimulating a surge in the levels of GnRH and subsequently FSH and LHrelease from the pituitary. This surge of gonadotropins induces thecompletion of follicular maturation and causes rupture of the dominantor Graafian follicle and discharge of the ovum some 16 to 24 hours afterthe LH peak. During the period following the preovulatory surge, serumestradiol levels temporarily decline and plasma progesterone levelsbegin to rise.

Following ovulation, the post-ovulatory ovarian follicle cells under theinfluence of LH are luteinized to form a corpus luteum—the luteal phase.The diagnostic markers of the luteal phase of the menstrual cycle arethe marked increase in progesterone secretion by the corpus luteum, andthe uterine transformation that occurs in response to progesterone.Associated with luteal progesterone production, there is a lesspronounced increase in serum estrogen levels. As progesterone andestrogens increase, LH and FSH decline throughout most of the lutealphase. Towards the end of the luteal phase, in a non-conceptivemenstrual cycle, the corpus luteum regresses and serum FSH levels beginto rise to initiate follicular growth for the next cycle.

FSH and LH are distinguished from each other by their ability tostimulate follicular development or ovulation, respectively. Both agentsare known to stimulate an increase in intracellular cAMP concentrations.Agents that mimic cAMP such as forskolin or stable analogs of cAMP havebeen shown, in vitro, to resemble the effects of FSH in granulosa cellsfrom immature follicles, and to resemble the effects of LH in cells frommature follicles. Although alternative intracellular pathways have beenproposed for both FSH and LH, it is well accepted that cAMP isstimulated in response to both gonadotropins. If and when elevations incAMP levels are associated with follicular development and maturation orovulation induction depends on the cell types and the presence orabsence of the respective receptors. Indeed, it has been demonstratedthat mice which are deficient in a particular phosphodiesterase haveimpaired ovulation and diminished sensitivity of granulosa cells togonadotropins.

Infertility treatments currently in clinical use incorporate some of theregulatory events described above. One agent which stimulates folliculargrowth and is used for treatment of anovulation is clomiphene.Clomiphene is a nonsteroidal antiestrogen that competes for estrogens attheir binding sites. It is thought that clomiphene binds to estrogenreceptors in the hypothalamus and pituitary and blocks the negativefeedback exerted by ovarian estrogens. The result is increased output ofgonadotropins (FSH and LH) during the early part of the follicularphase. The effect of clomiphene is to increase endogenous FSH serumlevels and to improve the growth and maturation of follicles.Subsequently either endogenous LH or exogenous LH/CG induce ovulation inthese patients.

In addition to clomiphene, women have been treated with ovulationinduction regimens which include commercial preparations of the humangonadotropins, including follicle stimulating hormone (FSH) andluteinizing hormone (LH) or chorionic gonadotropin (CG), all of whichwere first obtained by purification of urine from pregnant women andmore recently by recombinant technology. In general, this treatment ishighly effective in stimulating folliculogenesis and steroidogenesis.Complications of this treatment result from the fact that thesepreparations and regimens can over-stimulate follicular development andmaturation of follicles. In a subset of patients, the ovary can becomehyperstimulated, which may result in multiple ovulations and,consequently, multiple births. Not only can ovarian hyperstimulation belife threatening to the mother, it also typically results in newbornswith lower birth weight, who subsequently require intensive care. It isbelieved that the principal complications attributed togonadotropin-induced hyperstimulation and multiple pregnancies probablyresult from the prolonged effects of hCG. In addition, use ofgonadotropins in ovulation induction regimens can result in injectionsite reactions, both local and systemic. Consequently, the developmentof ovulation induction regimens using orally active agents with mildergonadotropin-like activity as opposed to therapies that use potentinjectables would be of substantial benefit. More importantly, it wouldbe a significant advantage if ovulation induction regimens could bedeveloped which result in less ovarian hyperstimulation and,consequently, present less danger to the mother and produce healthiernewborns.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a use of a non-peptide cAMPlevel modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the manufacture of a medicament for the induction ofovulation in a patient.

In a second aspect, the invention provides a use of a non-peptide cAMPlevel modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the induction of ovulation in a patient.

In a third aspect, the invention provides a use of a non-peptide cAMPlevel modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the manufacture of a medicament for the induction ofovulation in a patient, in a regimen whereby follicular maturation isinduced with clomiphene or an aromatase inhibitor, preferably anaromatase inhibitor selected from YM-511, Letrozole, Fadrozole, andAnastrozole, more preferably selected from Letrozole and Anastrozole,prior to ovulation induction.

In a fourth aspect, the invention provides a use of a non-peptide cAMPlevel modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for the induction of ovulation in a patient, in a regimenwhereby follicular maturation is induced with clomiphene or an aromataseinhibitor, preferably an aromatase inhibitor selected from YM-511,Letrozole, Fadrozole, and Anastrozole, more preferably selected fromLetrozole and Anastrozole, prior to ovulation induction.

In a fifth aspect, the invention provides a kit for use in inducingovulation, the kit comprising an ovulation inducing dose of anon-peptide cAMP level modulator, preferably a PDE inhibitor, morepreferably a PDE4 inhibitor, and instructions for its use in inducingovulation.

In a sixth aspect, the invention provides a kit for use in ovulationinduction and/or assisted reproductive technologies (ART), the kitcomprising sufficient daily doses of FSH and/or a compound having FSHeffect, preferably clomiphene or an aromatase inhibitor, more preferablyan aromatase inhibitor selected from YM-511, Letrozole, Fadrozole, andAnastrozole, to cause follicular maturation, and an ovulation inducingdose of a non-peptide cAMP level modulator, preferably a PDE inhibitor,more preferably a PDE4 inhibitor, and instructions for the use of thekit in ovulation induction or ART.

In a seventh aspect, the invention provides a use of a non-peptide cAMPlevel modulator, preferably a PDE inhibitor, more preferably a PDE4inhibitor, for inducing ovulation in a patient, in a regimen wherein hCGor LH are also administered to induce ovulation, and the hCG or LH areadministered at a reduced dose compared to the amount of hCG or LHnormally required to induce ovulation in the same patient.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of improving fertility in afemale host comprising the administration of a non-polypeptide cyclicadenosine monophosphate (cAMP) level modulator to the female host. Inanother aspect, the invention provides for specific administration ofthe non-polypeptide cAMP level modulator to induce ovulation of thedominant mature follicle prior to the luteal phase of an ovulatorycycle. Preferred non-polypeptide cAMP level modulators includephosphodiesterase inhibitors, particularly inhibitors ofphosphodiesterase 4 isoforms.

Although effects of PDE inhibitors on LH-stimulated steroidogenesis fromgranulosa cells, in vitro, have been reported, the present inventiondescribes two novel findings. First, the PDE inhibitors fail to enhanceFSH-stimulated follicular growth in vivo, despite the accepted role ofcAMP in both FSH and LH cellular pathways. Moreover, evidence ispresented that PDE inhibitors did enhance gonadotropin-stimulatedsteroidogenesis in vitro, which further exemplifies the novel activityof the PDE inhibitor on LH-dependent ovulation. Second, the PDEinhibitors increased the ovulation rate, in vivo, in the absence ofadded LH or hCG. Considering the oral activity of PDE inhibitors, thissecond finding provides the basis for the first potential injection-freeregimen for ovulation induction, since the PDE inhibitors can be used inconjunction with existing regimens as described below.

The invention also provides for the stimulation of folliculardevelopment prior to the administration of a non-polypeptide cAMP levelmodulator which comprises the administration of an agent which increasesFSH concentrations during the follicular phase of the host's ovulatorycycle. The objective of the invention in increasing FSH relates solelyto follicular development and maturation and not ovulation induction.Preferred agents include FSH, itself, clomiphene, selective estrogenreceptor modulators, aromatase inhibitors and selective modulators ofthe neuroendocrine regulation of FSH production.

In still another aspect, the invention provides for theco-administration of a non-polypeptide cAMP level modulator with LH orchorionic gonadotropin (CG) prior to the luteal phase of the femalehost's ovulatory cycle. Co-administration can occur sequentially orsimultaneously, as well as by the same or different modes of delivery(e.g., parenterally and or orally). In addition, the invention providesfor the use of lower concentrations of LH or CG administered to the hostthan concentrations that are used in current ovulation inductionregimens and thereby lowering the likelihood of ovarianhyperstimulation.

Additionally, the present invention provides for the use of anon-polypeptide cAMP level modulator as a therapeutic agent inreplacement of or to enhance the effect of hCG or LH in the collectionof oocytes for in vitro fertilization.

Thus, the invention provides for the use of a non-polypeptide cAMP levelmodulator as a small molecule therapeutic (e.g., phosphodiesteraseinhibitors) that is administered orally rather than by injection, therequired route of administration for proteins and the mode ofadministration in current ovulation induction regimens. Oraladministration avoids the acute and systemic side effects associatedwith such injections. Foremost, the small molecule therapeutic iseffective in inducing ovulations and can be administered alone or withor without LH or CG and alternatively, in lower concentrations of LH orCG than are currently used, and thus, lessen the occurrence of ovarianhyperstimulation and its associated risks. Consequently, multiple birthsand life threatening complications for the mother and newborns can beaverted. In addition, the present invention provides for the opportunityof earlier diagnostic testing for pregnancy than current ovulationinduction regimens involving the use of CG.

The treatment methods of the invention will be useful for treatment ofinfertility in humans, but also in other mammals (such as horses andlivestock e.g. cattle, sheep, cows and the like) and other species suchas piscine (i.e., fish) and avian (i.e., fowl).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a generalized ovulationinduction regimen in humans.

FIG. 2 is a bar graph representation of the effect of phosphodiesteraseinhibitors in vitro (i.e., Compound 1 and Compound 2) on increasingintracellular cAMP levels in granulosa cells.

FIG. 3 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on follicle maturation inrats, in vivo.

FIG. 4 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on ovulation with CG inrats, in vivo.

FIG. 5 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 1) on ovulation with andwithout CG in rats, in vivo.

FIG. 6 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 2) on ovulation with andwithout CG in rats, in vivo.

FIG. 7 is a bar graph representation of the effect of aphosphodiesterase inhibitor (i.e., Compound 2) on ovulation followingoral and subcutaneous administration.

FIG. 8 is a bar graph representation of the effect of PDE inhibitorCompound 3 on ovulation in vivo following oral and subcutaneousadministration.

FIG. 9 is a bar graph representation of the effect of PDE inhibitorCompound 4 on ovulation in the presence or absence of a subeffectivedose of hCG in vivo.

FIG. 10 is a bar graph representation of the effect of PDE inhibitorCompound 2 on ovulation and fertility.

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples and claims.

“Administration” refers to the delivery of a therapeutic agent into afemale host. In the context of the present invention, this would includethe delivery of a non-polypeptide cAMP level modulator and/or an agentwhich increases FSH concentrations. This term is more fully describedunder the section entitled, “Pharmaceutical Compositions” containedherein.

“Ovulation” for the purposes herein refers to the process where an ovumor ova are released from the ovaries. As midcycle approaches, there is adramatic rise in estrogen, followed by an LH and to a lesser extent anFSH surge. This triggers the dominant follicle to ovulate. Ovulationconsists of rapid follicular enlargement followed by protrusion of thefollicle from the surface of the ovarian cortex. Ultimately, rupture ofthe follicle results in the extrusion of an oocyte-cumulus complex. Theremnant of the dominant follicle then reorganizes to become the corpusluteum.

“Anovulation” refers to a lack of ovulation.

“Non-polypeptide cAMP level modulator” refers to compounds that are notcomposed of amino acids in their entirety, irrespective ofglycosylation, and act, directly or indirectly, to increaseintracellular levels of cAMP. Such compounds can increase cAMP levels bystimulating cAMP synthesis or by inhibiting its degradation, or both.Examples of modulators which increase the synthesis of cAMP includeactivators of adenyl cyclase such as forskolin. Examples of modulatorsthat decrease cAMP degradation include inhibitors of phosphodiesterasessuch theophylline.

“Female Host” means an individual of female gender of a species to whichagents are administered in accordance with the present invention. Humansother mammalians and other species such as fish and fowl are included bydefinition herein.

“Phosphodiesterase inhibitor” refers to chemical compounds which blockor inhibit phosphodiesterases (PDE's) whose action is to inactivatetheir cyclic nucleotide targets (i.e., cAMP and cGMP) by hydrolyticcleavage of the 3′-phosphodiester bond, resulting in passiveaccumulation of specific cyclic nucleotides. Inhibitors can benon-selective for all phosphodiesterase isoforms or selective forspecific isoforms. See compounds cited herein.

“Phosphodiesterase Isoforms” refers to a family of isozymes or isoformsresponsible for the metabolism or degradation of the intracellularsecond messengers, cAMP and cGMP. Specific isoforms can have highlyselective cellular and subcellular localizations. Examples ofphosphodiesterase isoforms include PDE3 and PDE4.

“Follicular Phase” refers to the first part of the menstrual cycle andis characterized by a progressive increase in circulating levels ofestradiol and inhibin B by the developing Graafian follicle.

“Prior to the Luteal Phase” refers to the period of the menstrual cyclebefore the shift from the estrogen dominated follicular phase to theprogesterone dominated luteal phase. Prior to the luteal phase, theestrogen levels are generally greater than or equal to 150pg/ml/follicle for a follicle of 16 mm in diameter and the follicle sizeis generally no less than 14 mm in diameter. These criteria are notabsolute and will vary from patient to patient. In the context of thepresent invention and in terms of the timing of administration of thenon-polypeptide cAMP level modulator, the non-polypeptide cAMP levelmodulator can be administered to a female host at the time point of anexisting ovulation induction regimen at which hCG or LH is normallyadministered to said host.

“Ovulatory Cycle” or “menstrual cycle” refer to a series of cyclicalevents over a species-specific period of time including folliculargrowth and development, recruitment, selection, dominance, ovulation,and corpus luteum formation and demise. Functionally, the cycle can bedivided into three phases, the follicular, the ovulatory and the lutealphases. This cycle can also be referred to the menstrual cycle.

“Ovulation Induction” refers to the process wherein a polypeptide(s)and/or synthetic chemical is used to bring about ovulation in femalehosts who are other wise anovulatory, resulting in induction offollicular rupture and ovulation of fertilizable oocytes. Ovulationinduction does not include the preceding events in time during theovulatory cycle of follicular maturation and development.

“Ovarian Hyperstimulation” refers to pharmacological intervention of anovulatory or anovulatory menstrual cycle. It entails maturation ofmultiple follicles resulting in codominance of numerous follicles andthe availability of multiple fertilizable oocytes.

“Follicle” refers to the fluid filled sac that surrounds the ovum, thesac also containing granulosa cells.

“Follicular Development” refers to the progressive growth anddevelopment of ovarian follicles, particularly during the follicularphase of the ovulatory cycle and leading to the recruitment anddominance of a follicle destined to ovulate.

“Follicle Stimulating Hormone (FSH) and isoforms” refers to a hormonereleased by the pituitary that stimulates the growth of ovarianfollicles and isoforms of FSH as described, for example, in U.S. Pat.No. 5,087,615, incorporated by reference herein.

“Selective Estrogen Receptor Modulators” refers to chemical compounds orpolypeptides that act as an estrogen receptor antagonist at the level ofthe hypothalamus and the pituitary gland, and as an agonist at the levelof the uterus. Examples of such modulators can include tamoxifen,raloxifene, toremifene, clomiphene and droloxifene. Reference,Endocrinology, 1999 December: 138(12): 5476-5484 is hereby incorporatedby reference.

“Aromatase Inhibitors” refer to chemical compounds or polypeptides thatblock or inhibit the activity of aromatase which is an enzyme thatconverts androgens to estrogens. Examples of aromatase inhibitorsinclude Letrozole, Anastrozole and Vorozole. References, 1) Journal ofEndocrinology, 2000 February: 164(2): 225-238; and 2) Journal of SteroidBiochemistry and Molecular Biology, 1997 April: 61(3-6): 157-166; arehereby incorporated by reference.

“Related Steroidogenic enzymes” refers to any enzyme that is involvedwith the catalysis of biochemical reactions leading to the synthesis ofestrogen and progesterone including 3-β-hydroxysteroid dehydrogenase andinhibitors of this enzyme include daidzein, genistein, biochanin A andformononetin. Reference, Journal of Steroid Biochemistry and MolecularBiology, 1996 April: 58(1): 95-101 is hereby incorporated by reference.

“Clomiphene” refers to2-[4-(2-chloro-1,2-diphenylethenyl)phenoxy]-N,N-diethylethanamine andits salts.

“Lutenizing Hormone” refers to a hormone released by the pituitary thatserves the dual purpose of causing a dominant follicle to release itsegg and stimulating the corpus luteum to secrete progesterone.

“Reduced concentrations” refers to lower concentrations of anadministered agent when compared to standard levels of administeredagents. In the context of the present invention, lower concentrations ofLH or CG are administered than are administered in existing ovulationinduction regimens.

“Existing Ovulation Induction Regimens” refers to current methods ofinducing ovulation including the use of clomiphene, gonadotropins (i.e.,FSH, LH and CG) or a combination of these agents to promotefolliculogenesis and induced ovulation in anovulatory females. Theregimens are varied in terms of the timing, frequency and concentrationof the agents administered. This definition includes modifications ofcurrent regimens which still require the administration of hCG or LH atsome time point during the ovulation induction regimen. The followingtreatises on female infertility, stimulated folliculogenesis andovulation induction are incorporated by reference herein: ReproductiveEndocrinology, Surgery, and Technology, Volumes 1 and 2; Editors: E. Y.Adashi, J. A. Rock and Z. Rosenwaks; Lippincott-Raven Publishers,Philadelphia, 1996 and Female Infertility Therapy Current Practice;Editors: Z. Shoham, C. M. Howles and H. S. Jacobs; Martin Dunitz Ltd,London, 1999.

“Chorionic Gonadotropin” refers to a glycoprotein hormone that isbiologically and immunologically similar to pituitary LH. In normalpregnancy, CG is produced by the placenta and can be used as adiagnostic marker of pregnancy by elevated levels in serumconcentration. The acronym hCG refers to human chorionic gonadotropin.

“Agent which increases FSH concentrations” refers to any composition ofmatter, protein or synthetic chemical, which when administered to afemale host increases the serum level concentrations of FSH, eitherdirectly or indirectly, by administering FSH itself, or an agent whichstimulates its endogenous production or inhibits its endogenousdegradation. The definition of this phrase and agent includes compoundswhich may not increase FSH concentrations but have follicle stimulatinghormone biological function and activity.

II. Principles of Ovulation Induction

Problems of inadequate or inappropriate gonadotropin levels have beenrecognized as causes of ovulatory dysfunction since the 1960s. Theclinical effectiveness of the various gonadotropin preparations used wasproportional to the amount of FSH administered. Initial evidencesuggested that exogenous LH is not required for adequatefolliculogenesis during ovulation induction. It became apparent,however, that women given only exogenous FSH fail to produce adequatefollicular estradiol for ovulation induction. The presence of at leastsome amount of exogenous or endogenous LH for ovulation induction in thehuman appears to be important. FIG. 1 illustrates a schematicrepresentation of a generalized ovulation induction regimen. An FSHpreparation is given at 75 IU/day for the first 7 days. At the end of 7days, an ultrasound scan is taken to assess follicular diameter andserum estradiol is measured. If the follicle is less than 12 mm, thedose of FSH is doubled, and a subsequent scan is taken in another 5-7days. Patients with follicles ≧15 mm diameter receive an ovulatory bolusdose of hCG.

III. Generalized Ovulation Induction Regimen

Ovulation induction is as much an art as a science. Despite a remarkablearray of treatment protocols, no single approach or specific method isuniquely correct. Certain principles do apply, however, and provide thebasis for safe and effective treatment. However, it should be noted thatthe criteria set forth below for inducing ovulation is given for examplepurposes only and may vary significantly by clinic, patient and the goalof the treatment.

The first cycle usually involves the administration of FSH dailybeginning on day 4 to 7 of a withdrawal bleed. Follicle growth andresponse are monitored by both estrogen levels and ultrasound. Adequatefollicle stimulation is usually achieved by 7 to 14 days of continuousFSH administration. Treatment with FSH for less than 8 days isassociated with increased spontaneous abortion rates among pregnantpatients.

Once sufficient follicle development has been achieved (two 16- to 18-mmfollicles together with a progressive rise in serum estrogen to 500 to1,000 pg/mL), hCG (5,000 or 10,000 IU) is administered. The timing ofhCG administration is important because the principal complicationsattributed to gonadotropin-induced hyperstimulation and multiplepregnancies probably result from the prolonged effects of hCG. Althoughthe half-life of hCG is approximately 8 hours, it may be detectable inthe patient's blood for 7 to 10 days after injection and misdiagnosed asa successful treatment pregnancy. Following administration of hCG, thecouple is instructed to have intercourse on that night and once or twicemore over the next 48 hours.

The fundamental rule regarding gonadotropin administration is that eachand every treatment cycle must be individualized, monitored, andadjusted appropriately. Monitoring is necessary not only to enhanceovulation and pregnancy rates but also to reduce the risk of severeovarian hyperstimulation and its potential consequences and multiplepregnancies.

This is achieved by frequent determinations of plasma/serum estradiollevels and by inspection of the ovaries by ultrasound. Estradiol levelsgenerally correlate with the number of growing follicles but notnecessarily with the number of mature follicles. Reliance on estradiollevels as the marker for follicle maturity may erroneously suggestfollicle maturity in the presence of multiple small follicles, resultingin premature hCG administration. Because follicular growth correlatesdirectly with ovum maturation, ultrasound assessment of mean folliculardiameter may be a better indicator in assessing maturity and timing ofhCG administration. Estrogen levels should therefore be used to assessearly follicular development as an indicator of gonadotropin response,and ultrasound should be used to assess the number and size of maturingfollicles.

The goal of most treatments is to maximize the potential for a singletonpregnancy while reducing the risk of hyperstimulation syndrome.Estradiol levels between 1000 and 1500 pg/mL appear to be optimal, butactual levels may vary depending on the laboratory used and thephysician's experience. The risk of hyperstimulation increases withhigher estradiol levels. In general, when serum estradiol exceeds 2000pg/mL, hCG should be administered with great caution or withheld toallow follicles to regress. In hypogonadotropic hypogonadism, the riskof severe hyperstimulation for values greater than 2400 pg/mL is 5% inpregnancy cycles and 1% in nonpregnant cycles. Furthermore, becausehyperstimulation tend to correlate with the number of follicles present,a decision to withhold hCG may be also based on an ultrasound finding of10 or more developing follicles.

The following treatises on female infertility, stimulatedfolliculogenesis and ovulation induction are incorporated by referenceherein: Reproductive Endocrinology, Surgery, and Technology, Volumes 1and 2; Editors: E. Y. Adashi, J. A. Rock and Z. Rosenwaks;Lippincott-Raven Publishers, Philadelphia, 1996 and Female InfertilityTherapy Current Practice; Editors: Z. Shoham, C. M. Howles and H. S.Jacobs; Martin Dunitz Ltd, London, 1999.

IV. Methods of the Present Invention

The present invention relates to a method of ovulation induction thatcomprises the administration of a non-polypeptide cAMP level modulatorin an ovulation induction regimen in which the modulator is administeredto enhance or substitute the administration of hCG (or LH), which istypically administered prior to the luteal phase of an induced ornaturally occurring ovulatory cycle. The modulator can be administeredalone or co-administered, either simultaneously or sequentially, withhCG (or LH), as well as by different modes of delivery (e.g.,parenterally or orally).

In another aspect, the invention provides for the co-administration,either simultaneously or sequentially, of a non-polypeptide cAMP levelmodulator with LH or CG prior to the luteal phase of the female host'sovulatory cycle. In addition, since it is believed that the principalcomplications attributed to gonadotropin induced ovarianhyperstimulation and consequent multiple pregnancies probably resultfrom the prolonged effects of hCG, the invention provides for the use oflower concentrations of LH or CG administered to the host thanconcentrations that are used in existing ovulation induction regimensand thereby lowering the likelihood of ovarian hyperstimulation, andconsequently averting the adverse effects associated with thatcondition: multiple births, low weight newborns and health complicationsfor the mother.

It should be noted that the administration of non-polypeptide cAMP levelmodulators have no therapeutic effect on follicular maturation anddevelopment during the ovulatory cycle.

Thus, the present invention relates to methods of inducing ovulation ina female host comprising the administration of a non-polypeptide cyclicadenosine monophosphate (cAMP) level modulator to the female host. Thenon-polypeptide cAMP level modulators act, directly or indirectly, toincrease intracellular levels of cAMP. Such compounds can increase cAMPlevels by stimulating cAMP synthesis or by inhibiting its degradation,or both. Examples of modulators which increase the synthesis of cAMPinclude activators of adenyl cyclase such as forskolin. Examples ofmodulators that decrease cAMP degradation include inhibitors ofphosphodiesterases such theophylline. Preferred non-polypeptide cAMPlevel modulators include phosphodiesterase inhibitors, particularlyinhibitors of phosphodiesterase 4 isoforms.

In still another aspect, the invention provides for specificadministration of the non-polypeptide cAMP level modulator prior to theluteal phase of the host's ovulatory cycle. As an enhancer or substituteof hCG in an ovulation induction regimen, in which hCG is typicallyadministered towards the end of the follicular phase but prior to theluteal phase of the ovulatory cycle.

The preferred timing of administration of the non-polypeptide cAMP levelmodulator is prior to the luteal phase of the host's ovulatory cycle.

The invention also provides for the stimulation of folliculardevelopment prior to the administration of a non-polypeptide cAMP levelmodulator for inducing ovulation which comprises the administration ofan agent which increases follicular stimulating hormone (FSH)concentrations during the follicular phase of the host's ovulatorycycle. Preferred agents include FSH, itself, clomiphene, selectiveestrogen receptor modulators, aromatase inhibitors and agents which arenot FSH but have FSH biological function and activity. Thus,administration of the agents described herein in a prescribed timingrelative to the growth and maturation of the follicle are claimed toimprove the process of ovulation and subsequent fertilization that musttake place if conception is to occur.

It should be noted that when a non-polypeptide cAMP level modulator isadministered alone and not co-administered with hCG, the presentinvention provides for the opportunity of earlier diagnostic testing forpregnancy than current ovulation induction regimens involving the use ofCG.

V. Phosphodiesterase Inhibitors

For phosphodiesterase inhibitors used as non-polypeptide cAMPmodulators, essentially any non-toxic inhibitor of PDE can be used inthe methods of the invention, including selective and non-selectiveinhibitors of PDE4. Suitable non-selective inhibitors of PDE4 andcombined PDE3/PDE4 inhibitors include theophylline(1,3-dimethyixanthine), isobutylmethylxanthine, AH-21-132(6-(4-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)-naphthyridine-bis(hydrogenmaleate), Org-30029(N′-hydroxy-5,6-dimethoxy-benzothiophene-2-carboximidamidehydrochloride), Org-20241(N-hydroxy-4-(3,4-dimethoxyphenyl)thiazole-2-carboximidamide), Org-9731(4fluoro-N-hydroxy-5,6-dimethoxybenzo(b)thiophene-2-carboximidamidemethanesulfonate), Zardaverine(6-[4-(difluoromethoxy)-3-methoxy-phenyl]-2H-pyridazin-3-one),vinpocetine (apovincaminic acid ethyl ester), EHNA (MEP-1)(9-(2-hydroxy-3-nonyl)adenine), Milrinone(6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile), Siguazodan(3-cyano-2-methyl-1-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine),Zaprinast(3-(2-propoxyphenyl)-2,4,7,8,9-pentazabicyclo[4.3.0]nona-1,3,6-trien-5-one),SK+F 96231 (2-(2-propoxyphenyl)-3,7-dihydropurin-6-one), Tolafentrine((−)-4′-(cis-1,2,3,4,4a,10b-Hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)naphthyridin-6-yl)-p-toluenesulfonanilide),Filaminast([1-(3-cyclopentyloxy-4-methoxy-phenyl)ethulideneamino]carbamate).

Particularly preferred are selective inhibitors specific for PDE4. Manyknown selective PDE4 inhibitors fall into one of six chemical structuralclasses, rolipram-like, xanthines, nitraquazones, benzofurans,naphthalenes and quinolines. Examples of rolipram-like analogs includeimidazolidinones and pyrrolizidinone mimetics of rolipram and Ro20-1724, as well as benzamide derivatives of rolipram such as RP 73401(Rhone-Poulenc Rorer). Xanthine analogs include Denbufylline (SmithKlineBeecham) and Arofylline (Almirall); Nitraquazone analogs includeCP-77,059 (Pfizer) and a series of pyrid[2,3d]pyridazin-5-ones (Syntex);Benzofuran analogs include EP-685479 (Bayer); Napthalene analogs includeT-440 (Tanabe Seiyaku); and Quinoline analogs include SDZ-ISQ-844(Novartis).

More preferred are the following:

Compounds disclosed in WO 97/42174 (Pfizer, Inc.):

wherein R¹ is cyclopentyl or cyclohexy, R² is ethyl, R³ is a carboxylicacid, ester or primary amide, hydroxymethyl or a carbonyl group, and R⁴is cyano;

Compounds disclosed in U.S. Pat. Nos. 5,710,160 and 5,710,170 (MerckFrost Canada, Inc.):

wherein R is selected from H, CO-(4-mepiperazinyl), CO-(pyrrolidinyl),CHNOH, 5-tetrazolyl, (CH)OHCH₃, COCH₃, CONHSO₂CH₃, CONHSO₂Ph,CONHSO₂CF₃, CONHSO₂C₆H₄CH₃(o), CH₂CONHSO₂Ph, CH₂CONHSO₂CF₃, COH(CF₃)₂and SO₂NHCOC₆H₄CH₃(o).

Compounds disclosed in WO 98/2007 (Darwin Discovery), represented by:

Compounds disclosed in WO 98/14432 (Janssen Pharm NV):

wherein R¹ is H or CH₃, R² is CH₃ or CHF₂, R³ is cyclopentyl, indanyl,cyclopropylmethyl, Ph(CH₂)₅, THF.

Compounds disclosed in WO 98/18796 (Novartis):

wherein X is NH₂, OH, NHPh, NPh₂, Ph, C₆H₄CO₂H and Y is Cl, CN, NO₂.

Compounds disclosed in WO 97/49702 (Pfizer, Inc.), represented by:

Compounds disclosed in WO 97/48697 (Rhône-Poulenc Rorer):

wherein R¹ is cyclopentyl, cyclopropyl, Ph(CH₂)₃, Benzyl or H, R² isCH₃, Benzyl, Tosyl or H, R³ is CH₃ or Cl, A is CH, C-alkyl, O or N, andB is C or N.

Compounds disclosed in WO 98/02440 (Bayer AG):

wherein R¹ is CH₃, OH, Oallyl, OCH₃, C₂H₅, Propyl or Acetyl, R² is 3-Cl,2,4-Cl₂, 3-NO₂, 3-Br, 4-F, 4-Cl, 2,4-(CH₃)₂ or 2,4(CH₃O)₂.

Compounds disclosed in WO 97/44337, WO 97/44036, WO 97/44322(Chiroscience Ltd.):

wherein R¹ is CH₃, Ethyl or Propyl, R² and R³ are independently selectedfrom H, optionally substituted aryl or heteroaryl, and R⁴ and R⁵ areindependently selected from acyl, alkyl (optionally substituted with OH,or halogen), aryl, heteroaryl, and acyl substituted with aryl orheteroaryl.

Compounds disclosed in U.S. Pat. No. 6,303,789 (Byk Gulden LombergChemische Fabrik GmbH):

wherein R¹ is 1-6C-alkoxy, 3-7C-cycloalkoxy, 3-7C-cycloalkylmethoxy,benzyloxy or 1-4C-alkoxy which is completely or predominantlysubstituted by fluorine; R² is hydrogen; and R³ is hydrogen; or R² andR³ are together a methylene group; R⁴ is hydrogen, 1-8C-alkyl,1-6C-alkoxy-1-4C-alkyl, 1-6C-alkylthio-1-4-alkyl,1-6C-alkylsulfinyl-1-4C-alkyl, 1-6C-alkylsulfonyl-1-4C-alkyl,1-8C-alkylcarbonyl, 3-7C-cycloalkyl, 3-7C-cycloalkymethyl,phenyl-1-4C-alkyl or 14C-alkyl which is completely or predominantlysubstituted by fluorine; R⁵ is phenyl, pyridyl, phenyl substituted byR⁵¹, R⁵² and R⁵³ or pyridyl substituted by R⁵⁴, R⁵⁵, R⁵⁶ and R⁵⁷,wherein R⁵¹ is hydroxyl, halogen, cyano, carboxyl, trifluoromethyl,1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, 1-4C-alkylcarbonyl,1-4C-alkylcarbonyloxy, amino, mono- or di-1-4C-alkylamino or1-4C-alkylcarbonylamino; R⁵² is hydrogen, hydroxyl, halogen, amino,trifluoromethyl, 1-4C-alkyl or 1-4C-alkoxy, R⁵³ is hydrogen, halogen,1-4C-alkyl or 1-4C-alkoxy, R⁵⁴ hydroxyl, halogen, cyano, carboxyl,1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl or amino; R⁵⁵ is hydrogen,halogen, amino or 1-4C-alkyl; R⁵⁶ is hydrogen or halogen; and R⁵⁷ ishydrogen or halogen; n is 1 or 2; m is 1 or 2; where the sum of m and nmay only assume the values 2 or 3, a salt of these compounds and theN-oxide of the pyridines or a salt thereof.

Compounds disclosed in U.S. Pat. No. 6,316,472 (Merck Frosst Canada):

or a pharmaceutically acceptable salt or hydrate thereof wherein:

-   Y represents N or N-oxide;-   y represents 0, 1 or 2;-   R¹ and R² are independently selected from:-   H, C₁₋₆ alkyl and haloC₁₋₆ alkyl,-   R³ and R⁴ are independently selected from H and C.sub.1-6 alkyl, or    R.sup.3 and R⁴ attached to the same carbon atom taken together    represent a carbonyl oxygen atom, or-   R³ and R⁴ attached to different carbon atoms considered in    combination with the carbon atoms to which they are attached along    with any intervening atoms and represent a saturated 5, 6- or    7-membered carbocyclic ring,-   R⁵ and R.sup.6 independently represent a member selected from the    group consisting of:-   H, C.sub.1-6 alkyl, haloC.sub.1-6 alkyl and CN;-   n represents an integer of from 0-6;-   Ar¹ is an aromatic group.

Particularly preferred are the following:

Rolipram (4-(3-cyclopentyloxy-4-methoxy-phenyl)pyrrolidin-2-one),theophylline (1,3-dimethylxanthine). Arofylline (Almirall), ARIFLO®(cis-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexane-1-carboxylicacid) (SmithKline Beecham), Roflumilast (Byk Gulden), Denbufylline(SmithKline Beecham), RS-17597 (Syntex), SDZ-ISQ-844 (Novartis),4-[2,3-bis(hydroxymethyl)-6,7-diethoxyn-aphthyl]-1-(2-hydroxyethyl)hydropyridin-2-one(T-440; Tanabe Seiyaku), methyl 3-[6-(2H-3,4,5,6tetrahydropyran-2-yloxy)-2-(3-thienylcarbonyl)ben-zo[b]furan-3-yl]propanoate(Bayer),2-methyl-1-[2-(methylethyl)(8-hydropyr-azolo[1,5-a]pyridin-3-yl)]propan-1-one(Ibudilast; Kyorin),N-(3,5-dichloro(4-pyridyl))(3-cyclopentyloxy-4-methoxyphenyl)carboxamide(RP 73401; Rhne-Poulenc Rorer),(1E)-1-aza-2-(3-cyclopentyloxy-4-methoxyp-henyl)prop-1-enyl aminooate(PDA-641; American Home Products),4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexanecarboxylic acid(SB207499; SmithKline Beecham), Cipamfylline (SmithKline Beecham),5-[3-((2S)bicyclo[2.2.1]hept-2-yloxy)-4-methoxyphenyl]-1,3-diazaperhydroi-n-2-one(CP-80633; Pfizer), 1-(3-nitrophenyl)-3-(3-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidine-2,4-dione (RS-25344; Syntex),4-((1R)-1-phenyl-2-(4-pyridyl)ethyl)-2-cyclopentyloxy-1-methoxybenzene(CDP-840; Celltech),(3-{[(3-cyclopentyloxy-4-methoxyphenyl)methyl]amino}-pyrazol-4-yl)methan-1-ol,Ro-20-1724 (Roche Holding AG), Piclamilast, Doxofylline (InstitutoBiologico Chemioterapico ABC SpA), RPR-132294 (Rhne-Poulenc Rorer),RPR-117658A (Rhne-Poulenc Rorer), L-787258 (Merck Frosst Canada), E-4021(Eisai Co. Ltd.), GF-248 (Glaxo Wellcome), SKF-107806 (SmithKlineBeecham), IPL-4088 (Inflazyme Pharmaceuticals Ltd.),{3-[(3-cyclopentyloxy-4-methoxyphenyl)methyl]-8-(methylethyl)purin-6-yl}ethylamine(V-11294A; Napp Research Centre Ltd.), Atizoram (Pfizer),5-(3-cyclopentyloxy-4-methoxyphenyl)pyridine-2-carboxamide (CP-353164;Pfizer), methyl3-[2,4-dioxo-3-benzyl-1,3-dihydropyridino[2,3-d]pyrimidin-yl]benzoate(CP-77059; Pfizer), CP-146523 (Pfizer), CP-293321 (Pfizer),CI-1044(Pfizer), PD-189659(Pfizer), CI-1018 (Pfizer), CP-220629(Pfizer),1-(3-nitrophenyl)-3-(4-pyridylmethyl)-1,3-dihydropyridino[2,3-d]pyrimidin-e-2,4-dione(RS-25344-000; Roche Bioscience), Mesopram (Schering AG),N-(2,5-dichloro(3-pyridyl))(8-methoxy(5-quinolyl))carboxamide (D-4418;Chiroscience), T-2585 (Tanabe Seiyaku),4-[4-methoxy-3-(5-phenylpentloxy-)phenyl]-2-methylbenzoic acid, XT-044(Hokuriku University), XT-611 (Kanzawa University), WAY-126120(Wyeth-Ayerst Pharmaceuticals Inc.),1-aza-10-(3-cyclopentyloxy-4-methoxyphenyl)7,8-dimethyl-3-oxaspiro[4.5]d-ec-7-en-2-one (WAY-122331 Wyeth-AyerstPharmaceuticals Inc.),[(3S)-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-methyl-5-oxopyrazolidinyl]-N-(3-pyridylmethyl)carboxamide(WAY-127093B; Wyeth-Ayerst Pharmaceuticals Inc.), PDB-093 (Wyeth-AyerstPharmaceuticals Inc.), 3-(1,3dioxobenzo[c]azolin-2-yl)-3-(3-cyclopentyloxy-4-methoxyphenyl)propanamide(CDC-801; Celgene Corp.), CC-7085 (Celgene Corp.), CDC-998 (CelgeneCorp.), NCS-613 (CNRS), CH-3697 (Chiroscience), CH-3442 (Chiroscience),CH-2874 (Chiroscience), CH-4139 (Chiroscience), RPR-114597 (Rhne-PoulencRorer), RPR-122818 (Rhne-Poulenc Rorer),(7aS,7R)-7-(3-cyclopentyloxy-4-m-ethoxyphenyl)-7a-methyl-2,5,6,7,7a-pentahydro-2-azapyrrolizin-3-one,GW-3600 (Glaxo-Wellcome), KF- 19514 (Kyowa Hakko Kogyo Co Ltd.), CH-422(Celltech Group), CH-673 (Celltech Group), CH-928 (Celltech Group),D-22888 (Asta Medica), AWD-12-232 (Asta Medica), YM-58997 (Yamanouchi).IC-485 (ICOS Corp.), KW-4490 (Kyowa Hakko Kogyo Co. Ltd.), YM-976(Yamanouchi), Sch-351591 (Celltech Group), AWD-12-343 (Asta Medica),N-(3,5-dichloro(4-pyridyl))-2-{1-[(4-fluorophenyl)methyl]-5-hydroxyindoli-n-3-yl}-2-oxoacetamide(AWD-12-281; Asta Medica), Ibudilast (Kyorin Pharmaceutical Co. Ltd.),Cilomilast (SmithKline Beecham), BAY-19-8004 (Bayer), methyl3-{2-[(4-chlorophenyl)carbonyl]-6-hydroxybenzo[b]furan-3-yl}propanoate,5-methyl-4-[(4-methylthiophenyl)carbonyl]-4-imidazolin-2-on-e,5,6-diethoxybenzo[b]thiophene-2-carboxylic acid (Tibenelast), and4-(3-bromophenyl)-1-ethyl-7-methylhydropyridino[2,3-b]pyridin-2-one(YM-58897; Yamanouchi).

In one embodiment, the invention provides for the use of a PDEinhibitor, preferably a PDE4 inhibitor, for triggering ovulation afterfollicular growth and maturation has been induced with FSH. Also withinthe scope of the invention is the use of a PDE inhibitor, particularly aPDE4 inhibitor, for triggering ovulation after follicular growth andmaturation has been induced with a compound or preparation having FSHactivity. A particularly preferred substitute for or adjuvant to FSHtreatment is an aromatase inhibitor, for example, YM-511 (Yamanouchi),Letrozole (Novartis), Anastrozole (AstraZeneca) or Fadrozole (Novartis).In a preferred regimen for assisted reproductive technologies (ART), inwhich it is desired to obtain multiple oocytes for in vitrofertilization, patients are administered an aromatase inhibitor (e.g. ator about 2.5-5 mg/day of Letrozole, or Anastrozole) from at or about day3 to at or about day 7, or from at or about day 3 to at or about day 8of the menstrual cycle, together with at or about 50-225, preferable50-150 IU FSH/day, starting on or about day 3 to day 7 of the menstrualcycle, FSH continuing until there are at least two leading follicleshaving a mean diameter of greater than or equal to at or about 16 mm. Atthis point, a PDE inhibitor, preferably a PDE4 inhibitor is administeredin a dose sufficient to trigger ovulation.

Alternatively, the aromatase inhibitor may be used as the sole folliclegrowth stimulating agent (i.e. in the absence of FSH), by using a higherdose of aromatase inhibitor (e.g. 2-10 mg/day of Letrozole orAnastrozole) and/or by prolonging administration (e.g. days 3 to 8, 3 to9, or 3 to 10). When follicular maturation is judged sufficient bysonography, an ovulation triggering dose of PDE inhibitor, preferablyPDE4 inhibitor is given. This regimen permits the collection of multipleoocytes, while avoiding injections, as all the agents used are orallyavailable.

In ovulation induction, it is desirable to cause the release of only oneovum. This can be achieved, according to the invention, using FSH tostimulate follicular growth and maturation, followed by administrationof a PDE inhibitor, preferably a PDE4 inhibitor, to trigger ovulation.Also within the scope of the invention are ovulation induction regimensin which follicular growth and maturation is induced with a substitutefor FSH, for example an aromatase inhibitor.

In a preferred regimen for ovulation induction, a patient isadministered a dose of aromatase inhibitor (e.g. 2.5-5 mg/day ofLetrozole or Anastrozole) from at or about day 3 to at or about day 7,or from at or about day 5 to at or about day 9 of the menstrual cycle(in the absence of FSH). Alternatively, a single dose of aromataseinhibitor may be given (e.g. 5-30 mg of Letrozole or Anastrozole,preferably 10 or 20 mg), at or about day 3 or day 4 of the menstrualcycle. Ovulation is triggered with an ovulation triggering dose of a PDEinhibitor preferably a PDE4 inhibitor. This regimen provides anovulation induction protocol that requires no injections.

VI. Pharmaceutical Compositions

The non-polypeptide cAMP level modulators and agents which increase FSHconcentrations in a female host (also referred to herein as “activecompounds”) of the invention, and derivatives, fragments, analogs andhomologs thereof, can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise theactive compounds and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, (e.g., intravenous, intradermal,subcutaneous), oral, inhalation, transdermal (topical), transmucosal,and rectal administration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The preferred route of administration for non-polypeptide cAMP levelmodulators including phosphodiesterase inhibitors, as well as forclomiphene, selective estrogen receptor modulators, aromatase inhibitorsand inhibitors of steroidogenic enzymes is by oral administration. Theseactive compounds also can be administered subcutaneously by injection,intravenously or trans-vaginally (for local administration). Thepreferred route of administration of FSH, LH or hCG is by subcutaneousinjection, but could also be administered intravenously.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal or vaginal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Compounds of the invention can be employed, either alone or incombination with one or more other therapeutic agents as discussedabove, as a pharmaceutical composition in mixture with conventionalexcipient, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for oral, parenteral, enteral or topicalapplication which do not deleteriously react with the active compoundsand are not deleterious to the recipient thereof. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines. See also Remington's Pharmaceutical Sciences. In general, asuitable effective dose of one or more compounds of the invention,particularly when using the more potent compound(s) of the invention,will be in the range of from 0.01 to 100 milligrams per kilogram ofbodyweight of recipient per day, preferably in the range of from 0.01 to20 milligrams per kilogram bodyweight of recipient per day, morepreferably in the range of 0.05 to 4 milligrams per kilogram bodyweightof recipient per day. The desired dose is suitably administered oncedaily, or several sub-doses, e.g. 2 to 4 sub-doses, are administered atappropriate intervals through the day, or other appropriate schedule.Such sub-doses may be administered as unit dosage forms, e.g.,containing from 0.05 to 10 milligrams of compound(s) of the invention,per unit dosage.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The following non-limiting examples are illustrative of the invention.

VII. EXEMPLIFICATION

An in vivo ovulation model was been developed in which FSH isadministered to immature rats bid×2 or 3 days to induce follicularmaturation, followed by a single ovulatory dose of hCG. A singleinjection of non-polypeptide cAMP level modulators (e.g., Compound 1,Compound 2, etc.) co-administered with a subovulatory dose of hCG orinjected alone resulted in an induction of ovulation. These results areconsistent with a model in which increased cAMP levels enhance orsubstitute for hCG but not FSH. The role of FSH in any ovulationinduction regimen is for promoting follicular development andmaturation, not ovulation induction.

The Compounds of Examples 1 through 9 are identified as follows:

-   Compound 1 is    Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylic    acid; Compound 2 is    3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide;    Compound 3 is 2-(4-(6,7-Diethoxy-2,3-bis(hydroxymethyl)    naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl) pthalazin-1 (2H)-one    hydrochloride; and Compound 4 is    7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.

Example 1 Effect of Compounds 1 and Compounds 2 on Rat Ovarian GranulosaCell cAMP Levels, Alone or with Gonadotropins In Vitro

Ovaries were removed from immature 25 day old, hypophysectomized,diethylstilbesterol treated Sprague-Dawley rats. The ovaries wererepeatedly punctured with 27 gauge needles to liberate granulosa cellsfrom the follicles. Cells were washed and re-suspended in McCoys 5Amedia+0.1% BSA+2 μM androstenedione. Viable cells in number of 100,000were loaded into 6-well tissue culture dishes in a 1.0 ml volume (withCompound 1 and Compound 2 at a concentration of 25 micromolar, eitheralone or in conjunction with a low, 0.1 pM dose of gonadotropin). Plateswere incubated in a 37° C. incubator, 100% humidity, 5.0% CO₂ for 48hrs. Conditioned media were assayed in a cAMP specific RIA. Results areexpressed as mean plus or minus standard deviations. As seen in FIG. 2,Compounds 1 and 2 cause a significant increase in cAMP levels in thepresence of subeffective concentrations of gonadotropin.

Example 2 Effect of the PDE Inhibitor Compound 1 on Follicle Maturation,In Vivo

Mature ovarian follicles generated in immature female rats by treatmentwith a sub-optimal dose of FSH (1.08 IU/rat/injection; bid×3 days) withand without co-administration of Compound 1 (0.1, 1, 10, and 50mg/kg/injection: bid×3 days). A single injection of an ovulatory dose ofhCG (20 IU) was administered with the final FSH injection. The PDEinhibitors were given in conjunction with sub-optimal doses of FSH. Allinjections were subcutaneously administered. Ovulation was determined 18hours after hCG administration by counting oocytes in oviduct. Resultsare expressed as mean plus or minus standard deviations. As seen in FIG.3, data represent average number of oocytes in oviducts of all rats ineach group and frequency of ovulating rats. As also noted in FIG. 3, aPDE inhibitor (Compound 1) inhibited (rather than stimulated) bothovulation administered at 50 mg/kg. The results demonstrate thatincreasing doses of PDE inhibitor failed to enhance the ability of asub-optimal dose of FSH to prepare follicles to ovulate.

Example 3 Effect of the PDE Inhibitor Compound 1 on Ovulation, in thePresence of a Subeffective Dose of hCG In Vivo

Mature ovarian follicles generated in immature female rats by treatmentwith an effective dose of FSH (2.16 IU/rat/injection; bid×2 days) wereinduced to ovulate with a single injection of hCG. hCG was administeredat a sub-effective dose (3 IU) with and without a single injection ofCompound 1 (50, 10 and 1 mg/kg) at the time of the final FSH injection.Ovulation was determined 18 hours after hCG administration by countingthe number of ova in the oviduct. As seen in FIG. 4, a single injectionof Compound 1 co-administered with a subovulatory dose of hCG resultedin an induction of ovulation. All injections were subcutaneous. Resultsare expressed as mean plus or minus standard deviations. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor enhances hCG-stimulated ovulation when a sub-optimal doseof hCG is administered. The effects of Compound 1, a known PDE inhibitorare shown.

Example 4 Effect of PDE Inhibitor Compound 1 on Ovulation in thePresence or Absence of a Subeffective Dose of hCG In Vivo

Following FSH induced follicular maturation (2.16 IU/rat/injection;bid×2 days) Compound 1 was injected with and without a sub-effectivedose of hCG. Ovulation was determined 18 hours after hCG/Compound 1administration by counting oocytes in oviduct. Data represent averagenumber of oocytes in oviducts of all rats in each group and frequency ofovulating rats. As seen in FIG. 5, a single injection of Compound 1administered alone, without a subovulatory dose of hCG, resulted in aninduction of ovulation in FSH pretreated rats. Results are expressed asmean plus or minus standard deviations. This data demonstrates that anon-polypeptide cAMP level modulator, in this case a PDE inhibitor,Compound 1, is able to induce ovulation in the absence of any injectedhCG. Previous experiments, and those presented here have shown thatfollicles prepared with these doses of FSH do not ovulate spontaneously,but require subsequent hCG administration.

Example 5 Effect of PDE Inhibitor Compound 2 on Ovulation in thePresence or Absence of a Subeffective Dose of hCG In Vivo

Following FSH induced follicular maturation (2.16 IU/rat/injection;bid×2 days) Compound 2 was injected with and without a sub-effectivedose of hCG. Ovulation was determined 18 hours after hCG/Compound 2administration by counting oocytes in oviduct. Data represent averagenumber of oocytes in oviducts of all rats in each group and frequency ofovulating rats. Results are expressed as mean plus or minus standarddeviations. As seen in FIG. 6, a single injection of Compound 2administered alone, without a subovulatory dose of hCG; resulted in aninduction of ovulation in FSH pretreated rats. This data demonstratesthat a non-polypeptide cAMP level modulator, in this case a PDEinhibitor, Compound 2, is able to induce ovulation in the absence of anyinjected hCG.

Example 6 Effect of PDE Inhibitor Compound 2 on Ovulation In VivoFollowing Oral and Subcutaneous Administration

Following FSH induced follicular maturation (2.16 IU/rat/injection;bid×2 days) Compound 2 was either injected subcutaneously (subcutis) oradministered by oral gavage. Ovulation was determined 18 hours afterCompound 2 administration by counting oocytes in oviduct. Data representaverage number of oocytes in oviducts of all rats in each group andfrequency of ovulating rats. Results are expressed as mean plus or minusstandard deviations. As seen in FIG. 7, administration of Compound 2 byeither subcutaneous or oral route resulted in an induction of ovulationin FSH pretreated rats. This data demonstrates that a non-polypeptidecAMP level modulator, in this case a PDE inhibitor, Compound 2, is ableto induce ovulation when administered orally.

Example 7 Effect of PDE Inhibitor Compound 3 on Ovulation In VivoFollowing Oral and Subcutaneous Administration

Following FSH induced follicular maturation (2.16 IU/rat/injection;bid×2 days) Compound 3 was either injected subcutaneously (subcutis) oradministered by oral gavage. Ovulation was determined 18 hours afterCompound 2 administration by counting oocytes in oviduct. Data representaverage number of oocytes in oviducts of all rats in each group andfrequency of ovulating rats. Results are expressed as mean plus or minusstandard deviations. As seen in FIG. 8, administration of Compound 3 byeither subcutaneous or oral route resulted in an induction of ovulationin FSH pretreated rats. This data demonstrates that a non-polypeptidecAMP level modulator, in this case a PDE inhibitor, Compound 3, is ableto induce ovulation when administered orally.

Example 8 Effect of PDE Inhibitor Compound 4 on Ovulation in thePresence or Absence of a Subeffective Dose of hCG In Vivo

Following FSH induced follicular maturation (2.16 IU/rat/injection;bid×2 days) Compound 4 was injected subcutaneously with and without asub-effective dose of hCG. Ovulation was determined 18 hours afterhCG/Compound 4 administration by counting oocytes in oviduct. Datarepresent average number of oocytes in oviducts of all rats in eachgroup and frequency of ovulating rats. As seen in FIG. 9, a singleinjection of Compound 4 administered with a subovulatory dose of hCGresulted in an induction of ovulation in FSH pretreated rats. Compound 4administered alone induced little or no ovulation. Results are expressedas mean plus or minus standard deviations. This data demonstrates that anon-polypeptide cAMP level modulator, in this case a PDE inhibitor,Compound 4, which is insufficient to induce ovulation alone, is able toinduce ovulation in the presence of a sub-effective dose of hCG.

Example 9 Effect of PDE Inhibitor Compound 2 on Ovulation and Fertility

Follicular maturation was induced in immature rats with FSH (4.33IU/rat/injection; bid×2 days) and Pregnant Mare's Serum Gonadotropin(PMSG) (1.5 IU/rat/injection; bid×2 days). This combination waspreviously found to induce follicular maturation and promote matingbehavior while maintaining a low spontaneous ovulation rate in theabsence of an additional injection of hCG. FSH and PMSG induced ratswere then treated with either Compound 2 (12 rats) or vehicle (12 rats)by subcutaneous administration. In one cohort of rats (6 rats pertreatment), ovulation was determined 18 hours after Compound 2 orvehicle administration by counting oocytes in oviduct. To assessfertility, another cohort of rats (6 rats per treatment) wereindividually placed overnight in a cage together with a single adultmale rat of proven fertility. The next day, male rats were removed andthe females were caged together by group until the day of parturition.The number of live full-term pups observed at the time of parturitionwas recorded. As seen in FIG. 10, a single injection of Compound 2resulted in an induction of ovulation in FSH/PMSG pretreated rats. Inaddition, rats treated with Compound #2 had an increased number of livepups at the time of parturition. Results for both ovulation and livepups are expressed as mean plus or minus standard deviations. This datademonstrates that a non-polypeptide cAMP level modulator, in this case aPDE inhibitor, Compound 2, induces ovulation of oocytes which arecapable of being fertilized in vivo.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The entire contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference.

1. A method of ovulation induction in a female host comprisingadministering to a female host in need thereof a non-polypeptide cAMPlevel modulator, wherein said non-polypeptide cAMP level modulator is aninhibitor of a phosphodiesterase 4 isoform selected from the groupconsisting of: Theophylline (1,3-dimethylxanthine),isobutylmethylxanthine, AH-21-132(6-(4-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)-naphthyridine-bis(hydrogenmaleate), Org-30029(N′-hydroxy-5,6-dimethoxy-benzothiophene-2-carboximidamidehydrochloride), Org-20241(N-hydroxy-4-(3,4-dimethoxyphenyl)thiazole-2-carboximidamide), Org-9731(4-fluoro-N-hydroxy-5,6-dimethoxybenzo(b)thiophene-2-carboximidamidemethanesulfonate), Zardaverine(6-[4-(difluoromethoxy)-3-methoxy-phenyl]-2H-pyridazin-3-one),vinpocetine (apovincaminic acid ethyl ester), EHNA (MEP-1)(9-(2-hydroxy-3-nonyl)adenine), Milrinone(6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile), Siguazodan(3-cyano-2-methyl-1-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine),Zaprinast(3-(2-propoxyphenyl)-2,4,7,8,9-pentazabicyclo[4.3.0]nona-1,3,6-trien-5-one),SK+F 96231 (2-(2-propoxyphenyl)-3,7-dihydropurin-6-one), Tolafentrine((−)-4′-(cis-1,2,3,4,4a,10b-Hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)naphthyridin-6-yl)-p-toluenesulfonanilide),Filaminast([1-(3-cyclopentyloxy-4-methoxy-phenyl)ethylideneamino]carbamate),Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylicacid;2-(4-(6,7-Diethoxy-2,3-bis(hydroxymethyl)naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl)pthalazin-1(2H)-one hydrochloride; and7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.
 2. A methodof ovulation induction in a female host comprising the administration ofa non-polypeptide cAMP level modulator to said host prior to the lutealphase of the host's ovulatory cycle, wherein said non-polypeptide cAMPlevel modulator is an inhibitor of a phosphodiesterase 4 isoformselected from the group consisting of: Rolipram(4-(3-cyclopentyloxy-4-methoxy-phenyl)pyrrolidin-2-one), theophylline(1,3-dimethylxanthine), isobutylmethylxanthine, AH-21-132(6-(4-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)-naphthyridine-bis(hydrogenmaleate), Org-30029(N′-hydroxy-5,6-dimethoxy-benzothiophene-2-carboximidamidehydrochloride), Org-20241(N-hydroxy-4-(3,4-dimethoxyphenyl)thiazole-2-carboximidamide), Org-9731(4-fluoro-N-hydroxy-5,6-dimethoxybenzo(b)thiophene-2-carboximidamidemethanesulfonate), Zardaverine(6-[4-(difluoromethoxy)-3-methoxy-phenyl]-2H-pyridazin-3-one),vinpocetine (apovincaminic acid ethyl ester), EHNA (MEP-1)(9-(2-hydroxy-3-nonyl)adenine), Milrinone(6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile), Siguazodan(3-cyano-2-methyl-1-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine),Zaprinast(3-(2-propoxyphenyl)-2,4,7,8,9-pentazabicyclo[4.3.0]nona-1,3,6-trien-5-one),SK+F 96231 (2-(2-propoxyphenyl)-3,7-dihydropurin-6-one), Tolafentrine((−)-4′-(cis-1,2,3,4,4a,10b-Hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)naphthyridin-6-yl)-p-toluenesulfonanilide),Filaminast([1-(3-cyclopentyloxy-4-methoxy-phenyl)ethylideneamino]carbamate),Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylicacid;2-(4-(6,7-Diethoxy-2,3-bis(hydroxymethyl)naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl)pthalazin-1(2H)-one hydrochloride; and7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.
 3. A methodof a combined treatment for stimulating follicular development andovulation induction in a female host comprising the administration of anagent which increases follicle stimulating hormone concentrations insaid host during the follicular phase of the host's ovulatory cycle andadministering a non-polypeptide cAMP level modulator to said host priorto the luteal phase of the host's ovulatory cycle, wherein saidnon-polypeptide cAMP level modulator is an inhibitor of aphosphodiesterase 4 isoform selected from the group consisting of:Rolipram (4-(3-cyclopentyloxy-4-methoxy-phenyl)pyrrolidin-2-one),theophylline (1,3-dimethyixanthine), isobutylmethyixanthine, AH-21-132(6-(4-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)-naphthyridine-bis(hydrogenmaleate), Org-30029(N′-hydroxy-5,6-dimethoxy-benzothiophene-2-carboximidamidehydrochloride), Org-20241(N-hydroxy-4-(3,4-dimethoxyphenyl)thiazole-2-carboximidamide), Org-9731(4-fluoro-N-hydroxy-5,6-dimethoxybenzo(b)thiophene-2-carboximidamidemethanesulfonate), Zardaverine(6-[4-(difluoromethoxy)-3-methoxy-phenyl]-2H-pyridazin-3-one),vinpocetine (apovincaminic acid ethyl ester), EHNA (MEP-1)(9-(2-hydroxy-3-nonyl)adenine), Milrinone(6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile), Siguazodan(3-cyano-2-methyl-1-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine),Zaprinast(3-(2-propoxyphenyl)-2,4,7,8,9-pentazabicyclo[4.3.0]nona-1,3,6-trien-5-one),SK+F 96231 (2-(2-propoxyphenyl)-3,7-dihydropurin-6-one), Tolafentrine((−)-4′-(cis-1,2,3,4,4a,10b-Hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)naphthyridin-6-yl)-p-toluenesulfonanilide),Filaminast([1-(3-cyclopentyloxy-4-methoxy-phenyl)ethylideneamino]carbamate),Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylicacid;2-(4-(6,7-Diethoxy-2,3-bis(hydroxymethyl)naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl)pthalazin-1(2H)-one hydrochloride; and7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.
 4. A methodof claim 3 wherein said agent is follicle stimulating hormone.
 5. Amethod of claim 3 wherein said agent is clomiphene.
 6. A method of claim3 wherein said agent is a selective estrogen receptor modulator.
 7. Amethod of claim 3 wherein said agent is an aromatase inhibitor.
 8. Amethod of claim 3 wherein said agent is an inhibitor of relatedsteroidogenic enzymes that results in a decrease in total estrogenproduction.
 9. A method of claim 3 wherein luteinizing hormone (LH) isalso administered to said host to induce ovulation prior to the lutealphase of the host's ovulatory cycle.
 10. A method of claim 3 wherein LHis also administered at reduced concentrations compared to existingregimens to said host to induce ovulation prior to the luteal phase ofthe host's ovulatory cycle.
 11. A method of claim 3 wherein chorionicgonadotropin is also administered to said host to induce ovulation priorto the luteal phase of the host's ovulatory cycle.
 12. A method of claim3 wherein chorionic gonadotropin is also administered at reducedconcentrations compared to existing regimens to said host to induceovulation prior to the luteal phase of the host's ovulatory cycle.
 13. Amethod of ovulation induction in a female host comprising theadministration of a non-polypeptide cAMP level modulator to said host atthe time point of an existing ovulation induction regimen at which humanchorionic gonadotropin (hCG) or LH is administered to said host, whereinsaid non-polypeptide cAMP level modulator is an inhibitor of aphosphodiesterase 4 isoform selected from the group consisting of:Rolipram (4-(3-cyclopentyloxy-4-methoxy-phenyl)pyrrolidin-2-one),theophylline (1,3-dimethylxanthine), isobutylmethylxanthine, AH-21-132(6-(4-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)-naphthyridine-bis(hydrogenmaleate), Org-30029(N′-hydroxy-5,6-dimethoxy-benzothiophene-2-carboximidamidehydrochloride), Org-20241(N-hydroxy-4-(3,4-dimethoxyphenyl)thiazole-2-carboximidamide), Org-9731(4-fluoro-N-hydroxy-5,6-dimethoxybenzo(b)thiophene-2-carboximidamidemethanesulfonate), Zardaverine(6-[4-(difluoromethoxy)-3-methoxy-phenyl]-2H-pyridazin-3-one),vinpocetine (apovincaminic acid ethyl ester), EHNA (MEP-1)(9-(2-hydroxy-3-nonyl)adenine), Milrinone(6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile), Siguazodan(3-cyano-2-methyl-1-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine),Zaprinast(3-(2-propoxyphenyl)-2,4,7,8,9-pentazabicyclo[4.3.0]nona-1,3,6-trien-5-one),SK+F 96231 (2-(2-propoxyphenyl)-3,7-dihydropurin-6-one), Tolafentrine((−)-4′-(cis-1,2,3,4,4a,10b-Hexahydro-8,9-dimethoxy-2-methylbenzo(c)(1,6)naphthyridin-6-yl)-p-toluenesulfonanilide),Filaminast([1-(3-cyclopentyloxy-4-methoxy-phenyl)ethylideneamino]carbamate),Cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylicacid;2-(4-(6,7-Diethoxy-2,3-bis(hydroxymethyl)naphthalen-1-yl)pyridin-2-yl)-4-(3-pyridyl)pthalazin-1(2H)-one hydrochloride; and7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine.
 14. A methodof claim 13 wherein the non-polypeptide cAMP level modulator isco-administered with hCG or LH.
 15. A method of claim 13 wherein thenon-polypeptide cAMP level modulator is administered alone and notco-administered with hCG or LH.